Control material and methods for cell analyzers

ABSTRACT

This disclosure relates to verifying the operation of cell analyzers, including microscope-based cell imaging and counting analyzers. In one general aspect, a mixture of micro-beads having known characteristics is introduced into the analyzer. One or more images of the mixture are acquired with the analyzer&#39;s microscope, the images are analyzed, and a determination is made about whether results meet one or more predetermined quality control thresholds. Also disclosed is a hematology control material that can be used to perform the verification and includes a solvent, a dye dissolved in the solvent, and micro-beads suspended in the solvent. In another general aspect, a quality control method for the analyzers includes capturing images of samples that include patient cells using at least a microscope, extracting sample-specific information about properties of the patient samples from the images, and testing information from the samples against predetermined standards to verify the operation of the analyzer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.62/586,650, which was filed on Nov. 15, 2017, and is herein incorporatedby reference.

This application also relates to automated microscopic cell analysis andrelated technology described in US published application number20170328924, which was published on Nov. 16, 2017, and PCT publishedapplication number WO2018/009920, which was published on Jan. 11, 2018.Both of these applications are herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to Quality Control (QC) materials andcalibrators and associated methods for microscopy-based and traditionalcell imaging and counting systems.

BACKGROUND OF THE INVENTION

Complete Blood Count (CBC) control materials have been developed forcellular impedance and flow cytometry analyzers (traditional CBCsystems). These traditional CBC systems generally consist of complexfluidic channels that require regular maintenance, calibration, andcontrol accuracy. The risks of drifts and erroneous results are commondue to clogs, partial blockages, and protein or salt buildup. A qualitycontrol material is therefore used to verify calibration, check forsystem failure, and demonstrate the proficiency of the operator.Traditional hematology control materials are made of stabilized bloodfrom human and animal sources. The blood is stabilized by use offixatives or preservatives so that it will not degrade to the pointwhere cells can no longer be analyzed. This use of stabilized blood is acompromise to provide traditional CBC systems with a method ofperforming quality control on a regular basis.

Traditional CBC systems rely on the metaproperties of cells, such as DCconductivity, RF impedance, light scatter, and light extinction to countand classify cells. Counting and classifying cells based on thesemetaproperties has been determined empirically and validated throughyears of development and use of traditional CBC analyzers. It is throughthis use of metaproperties that stabilized control materials cansimulate a fresh human blood sample. For example, avian red blood cells(RBC) can be used to simulate human lymphocytes. Under a microscope, thecells are clearly nucleated RBCs, but because the metaproperties aresimilar to those of human lymphocytes, the avian cells can be used as asurrogate. Preservatives and stabilizing agents are added to the bloodto mitigate degradation of the cells in their natural matrix.

Often the metaproperties of stabilized blood controls do not exactlymatch those of fresh, human whole blood. The stabilization process canresult in a significant alteration of size, shape, and granularity ofthe cells. For this reason, the software gate parameters used forcounting and classifying controls on traditional CBC systems aregenerally different than those used for whole blood. These specializedgate parameters adjust for shifts between fresh human blood andstabilized blood control materials. In most cases, if one was to run acontrol material in blood sample mode, the results would not align withthe provided insert ranges. Conversely, if a fresh blood is run incontrol mode, the results would also be erroneous.

Stabilized blood controls generally require special handling and storageand have a short shelf life. They generally are shipped and stored atrefrigerated temperatures (2-8° C.) and are shipped overnight to ensuresafe delivery. Once the control has been first used, it starts todegrade and can typically only be used for 1-2 weeks (open-containerstability). Due to the short closed-container shelf life of typicallyless than 90 days, it is common for control manufacturers to requirethat laboratories issue standing orders to ensure that they can supplysufficient material to their customers. If a laboratory is not using astanding order or if it has used more than it has planned, it can bedifficult and sometimes impossible to obtain control materials due toproduction schedule. These requirements can put a strain on laboratoriesand clinics that are not able to keep up with strict planningrequirements. Also, these controls are expensive when compared to thosefor other common clinical diagnostic tests, such as clinical chemistry,blood gas, and electrolytes.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one general aspect, the invention features a method of verifying theoperation of a microscope-based cell imaging and counting analyzer. Themethod includes providing a mixture of micro-beads having knowncharacteristics, introducing the mixture into the analyzer, acquiringone or more images of the mixture with the analyzer's microscope,analyzing the mixture of micro-beads in the images, and determiningwhether results of the analyzing meet one or more predetermined qualitycontrol thresholds.

In preferred embodiments, the mixture of beads can have a knownconcentration. The mixture of beads can be counted by image processinglogic associated with the analyzer. The mixture of beads can beclassified by image processing logic associated with the analyzeraccording to their different characteristics. The introducing themixture can include introducing the mixture into a test cartridge andintroducing the test cartridge into the analyzer. The providing canprovide a mixture of micro-beads that includes at least some fluorescentmicro-beads. The providing can provide a mixture of micro-beads ofdifferent sizes. The determining can include determining a distributionof bead sizes. The determining can include determining whether amicro-bead count meets a predetermined accuracy standard. The micro-beadcount can be used for calibration of the system. The mixture can bestained and the method can further include determining whether theanalyzer can detect the one or more properties of the stain within oneor more predetermined quality control thresholds. The method can furtherinclude performing internal checks on patient samples.

In another general aspect, the invention features a microscope-basedcell imaging and counting analyzer. The analyzer includes a microscopeoperative to acquire one or more images of a mixture of micro-beads,image processing logic responsive to the microscope and operative toanalyze image characteristics of the mixture of micro-beads in theimages, and quality control decision logic responsive to the imageprocessing logic and operative to determine whether the imagecharacteristics of the images of the micro-beads meet one or morepredetermined quality control thresholds.

In a further general aspect, the invention features a hematology controlmaterial that includes a solvent, a dye dissolved in the solvent, andmicro-beads suspended in the solvent. In preferred embodiments themicro-beads can include different sizes of micro-beads. The micro-beadscan include at least some fluorescent micro-beads. At least some of thebeads can be on the order of the size of at least one type of bloodcells. At least some of the beads can be chosen to simulate platelets,red blood cells, white blood cells, and/or reticulocytes. At least someof the beads can be about in the range of 1-20 micrometers in diameter.The beads can be made of silica or polystyrene. The beads can be ofdifferent colors and/or shapes. The beads can be used to simulate awhite cell differential. The beads can be in a concentration similar tohuman blood cells. The dye can simulate the absorbance of hemoglobinconcentration of blood at a minimum of one wavelength of light. Thebeads can be in a concentration similar to abnormal human blood cellconcentrations. The material can include a plurality of lots of beadseach with bead concentrations at different levels to simulate normal andabnormal human blood cell concentrations.

In another general aspect, the invention features a quality controlmethod for a microscopy-based cell imaging and counting analyzer. Themethod includes capturing images of samples that include patient cellsusing at least a microscope, extracting sample-specific informationabout properties of the patient samples from the captured images, andtesting information from the samples against predetermined standards toverify the operation of the analyzer.

In preferred embodiments the testing can test information from thepatient sample images captured by the microscope. The testing caninclude monitoring mean fluorescent intensity of cells by comparing thevalue to a pre-determined acceptable level. The testing can includecomparing red blood cells in the images to a pre-determined set offeatures to verify that the red blood cells have been properly sphered.The testing can include comparing red blood cells in the images to apre-determined roundness standard to verify that the red blood cellshave been properly sphered. The testing can include comparing red bloodcells in the images to a pre-determined secondary ring standard toverify that the red blood cells have been properly sphered. The testingcan include using image processing to check for clots and microbubbles.The testing can include checking that the cells are not overlapping. Themethod can further include rejecting a sample and notifying a user basedon invalid results of the step of testing. The capturing of patientsample images can be performed by a microscope and further includingcapturing further images of the patient samples with one or moreadditional cameras. The testing can use at least one macroscopic viewcamera to verify that an imaging region used for analysis is free frombubbles or voids. The testing can use at least one macroscopic viewcamera to calculate the area occupied by bubbles or voids in the imagingregion that is used for analysis. The testing can use at least onemacroscopic view camera to determine concentration gradients throughoutthe imaging region. The testing can use at least one macroscopic viewcamera to verify sample processing steps for diluting and mixing thepatient sample with a diluent have been performed without error.

Materials and methods according to the invention can provide bettersolutions for quality control for microscopy-based cell imaging andcounting systems, which can use different characteristics of cells thantraditional CBC systems for counting and classification.

Benefits of materials according to the invention can include:

-   -   Improved operator and system control for microscopy-based cell        imaging systems as compared to traditional CBC QC materials    -   Specific design for use with a microscopy-based cell imaging        systems    -   Extensive procedural and system controls for every test    -   No need for cold-chain (2-8° C.) shipping or refrigerated        storage    -   Longer shelf-life as compared to traditional CBC QC materials    -   Lower costs than traditional CBC QC materials

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cut-away view of an illustrative cell analyzer showinginternal components with a test cartridge being inserted;

FIG. 2 is a flowchart illustrating the operation of the cell analyzer ofFIG. 1;

FIG. 3A is a schematic image of an illustrative quality control materialthat can be used with cell analyzers, such as the microscope-basedanalyzer of FIG. 1;

FIG. 3B is a set of bright-field and fluorescent microscopic images ofmicroparticles used to simulate various cell types for use in a qualitycontrol material;

FIG. 3C is a microscopic image of a correctly sphered red blood cell;

FIG. 3D is a microscopic image of an incorrectly sphered red blood cellthat fails a roundness specification;

FIG. 3E is a microscopic image of a completely unsphered red blood cellthat fails specification because of a secondary ring detected in theimage;

FIG. 4 is a flowchart of an illustrative calibration method for the acell analyzer, such as the microscope-based analyzer of FIG. 1;

FIG. 5 is a flowchart of an illustrative method of verifying theoperation of a cell analyzer, such as the microscope-based analyzer ofFIG. 1;

FIG. 6 is a flowchart of an illustrative quality control method that canbe used with cell analyzers, such as the microscope-based analyzer ofFIG. 1;

FIG. 7 is an illustrative plot of mean fluorescence intensity againsttime for eight illustrative sample runs with an acceptable excursionlevel for the mean White Blood Cell (WBC) fluorescence signal during ameasurement performed by a cell analyzer, such as the microscope-basedanalyzer of FIG. 1;

FIG. 8A is a plan view of an illustrative test cartridge showing asample of whole blood deposited in the input port;

FIG. 8B is a plan view of the test cartridge of FIG. 8A showing initialmovement of the sample and reagent with the rotary valve in the firstopen position;

FIG. 8C is a plan view of the test cartridge of FIG. 8B with the valvein the second open position;

FIG. 8D is a plan view of the test cartridge of FIG. 8C illustrating thesample and the reagent in the imaging chamber;

FIG. 8E is a plan view of the test cartridge of FIG. 8D illustrating thesample and most of the reagent positioned in the mixing chamber; and

FIG. 8F is a plan view of the test cartridge of FIG. 8E illustrating allof the sample and the reagent positioned in the imaging chamber and thevalve in a final position.

DETAILED DESCRIPTION OF AN ILLUSTRATIVE EMBODIMENT I. Analyzer Structureand Operation

FIG. 1 is a cut-away view of an illustrative cell analyzer 200 with testcartridge 100 positioned so that the operator can introduce it into theanalyzer. The structure and operation of this analyzer are discussed inmore detail in the above-referenced published applications. From theoutside of the cell analyzer 200, one can see the housing 206, auser-interface screen 208, a printer 212, and a cartridge loading door270. When the cartridge loading door 270 is opened, the test cartridge100 can be placed on a cradle 220 of x-y stage, configured to receivetest cartridge 100 from the user. The cradle 220 provides mechanicalalignment of the cartridge to facilitate connections that are madebetween the analyzer and the cartridge. For example, a mechanicalpresser foot 230 may be placed in contact with a flexible surface on thetest cartridge to provide mechanical pressure onto packaged, on-boardreagents.

A valve driver 235 can be positioned to operate a rotary valve on thetest cartridge. A vacuum/pressure pump 240 supplies negative or positivepressure to a manifold 245, which interfaces with the test cartridge 100when it is placed in the cell analyzer as described below. The cellanalyzer 200 further includes system controller 250 to control movementof the fluids in the test cartridge by activating the vacuum/pressurepump 240, moving the mechanical presser foot 230, or operating the valvedriver 235 according to pre-programmed sequences. A monitoring camera255, positioned to acquire digital images of the fluids in thecartridge, provides feedback for the system controller 250. A monitoringlight source 256 may be a ring illuminator that surrounds the lens ofthe monitoring camera 255. Information from the monitoring camera 255 isused to provide feedback for controlling movement of liquids, forpositioning the rotary valve, and for confirming critical steps, asdiscussed in more detail below.

In this embodiment, a single monitoring camera is provided in a positionbelow the test cartridge, but one or more additional monitoring camerascan also be used. In another embodiment, for example, two monitoringcameras are included in the cell analyzer, with one positioned below thetest cartridge position facing upward, and another positioned above testcartridge facing downward. These are both provided with lowermagnification than the analyzer's microscope so they can monitor largerareas.

Also shown in FIG. 1 are the components that comprise the automatedmicroscope of the cell analyzer 200. At the base of the analyzer,bright-field light source 260 provides illumination through the testcartridge to the objective lens 265, operatively coupled to focusingmechanism 267. At the top of the analyzer, fluorescent light source 270provides illumination through dichroic mirror 277 to provide fluorescentexcitation of the sample. At the rear of the analyzer, digital camera280 captures images of the cells in the test cartridge 100 and transmitsthem to image processor/computer 290. In some embodiments, the cellanalyzer may further include a photometric light source 293 andphotometric detector 295 for measuring light transmission at one ormultiple wavelengths in a chamber in test cartridge 100, such as formeasuring hemoglobin, as is more fully explained below.

Turning our attention to FIG. 2 we will now describe the overalloperation of cell analyzer 200 configured to provide a “CBC withDifferential” analysis with reference to the test cartridge 401illustrated in FIGS. 8A-8F and cell analyzer 200 illustrated in FIG. 1.Prior to adding the blood sample from a patient presented at box 500 tothe test cartridge, the user first obtains a new test cartridge 401 atbox 505 and opens it to expose the input port 407. Blood from a fingerprick is applied to an input port 407 on the cartridge (see FIG. 8A) atbox 510 and the input port 407 is covered. The user inserts the testcartridge into the cell analyzer 200 at box 515. The test cartridge ismoved into the analyzer where mechanical and fluid connections are madebetween the analyzer and the cartridge as described above with referenceto FIG. 1. As a first step of analysis, the sample is drawn into themetering chamber passing through and into a photometric chamber 409(FIG. 8A). Absorbance of the blood is measured at box 520. Data fromabsorbance measurements are used to determine hemoglobin concentration.At box 530 sample in the metering chamber 483 is imaged using monitoringcamera 255 and analyzed to confirm that the metering chamber wasproperly filled at box 535. If an error is detected the analysis isterminated at box 537 and the user is alerted to the error andinstructed to remove the cartridge and reject the test.

If the pass-through conduit 413 is correctly filled the diluent/reagentchannel is primed at box 540 as described above with reference to FIG.8B. Rotary valve 415 is then turned to the position shown in FIG. 8C toisolate the sample and to allow diluent/reagent to wash the meteredvolume of blood out of the pass-through conduit 413 at box 545 whilebeing imaged by monitoring camera 255. The transfer continues until themonitoring camera 255 confirms that diluent/reagent plus sample hasalmost filled the imaging chamber as illustrated in FIG. 8D.

Once a sufficient volume of diluent/reagent is transferred, rotary valve415 is positioned as shown in FIG. 8E and the total volume of sample anddiluent/reagent is mixed, 550. At box 555 the entire volume 495 istransferred to the imaging chamber and rotary valve 415 is positioned asshown in FIG. 8F. Note that by transferring the entire volume of mixedsample 495, all of the metered volume of blood from the original sampleplus the unmetered volume of diluent/reagent is positioned in theimaging chamber at box 555.

If test cartridge 400 is used, it is inserted into cell analyzer 200 andanalysis begins at step 560. Analysis of test cartridge 401 or 402continues at step 560 when the x-y stage 225 moves the test cartridge401 to obtain bright-field and fluorescent images of the entire imagingchamber 403 at box 560. In an alternate embodiment, objective lens 265and/or digital camera 280 are moved and test cartridge 401 remainsstationary. In yet another embodiment objective lens 265 has sufficientfield of view to capture the entire imaging chamber 403 withoutmovement. Two digital images of each physical frame of the imagingchamber are transferred to image processor/computer 290 at box 565. Oneimage, taken with bright-field optics, can be compared to the otherimage taken with fluorescent optics to identify red blood cells, whiteblood cells and platelets. Further analysis of the white cell sizes andinternal structure can identify sub-types of white cells using patternrecognition.

At box 570 comparison of the bright-field and fluorescent images candifferentiate mature red cells from reticulocytes and nucleated redblood cells. By dividing each cell count by the known volume of themetering chamber 483, the concentration (cells per unit volume) can bedetermined. By using a sphering agent the planar sizes of red cells canbe transformed into mean corpuscular volume (MCV). Combining the redblood cell count with MCV and the volume of the metering chamber 483allows the calculation of hematocrit (HCT) and red cell distributionwidth (RDW). Further calculations using the separately measured HGB frombox 525, combined with the RBC count gives mean corpuscular hemoglobin(MCH), and mean corpuscular hemoglobin content (MCHC).

At box 575 the measured results are compared with previously definedlimits and ranges for the particular patient population anddetermination is made whether the results are within or outside normalexpected ranges. According to this determination results within normalranges are reported in box 580 and results that are outside the normalranges are reported in box 585. As will be discussed in more detailbelow, the cell analyzer 200 can also perform a variety of other qualitycontrol and calibration operations to ensure the accuracy of its resultsusing the microscope and/or the monitoring camera(s).

II. Control Material

Referring to FIG. 3, some of the quality control and calibrationoperations employ a surrogate control material. An illustrativesurrogate control material 600 can consist of a dye 602 and beads (e.g.,604, 606, 608, 610, 612) of various sizes, shapes, and/or colors in thedye. The dye is chosen such that the concentration and absorbancesimulates whole blood at at least one wavelength of light. The beads maybe made of any suitable material, including silica, polystyrene, orother polymer in composition. The beads could also be coated with ametal such as gold or silver. The sizes of the beads are typically inthe range of 1-20 micrometers to simulate the size of platelets, redblood cells, reticulocytes, and white blood cells. Some or all of thebeads may be fluorescent to aid in detection. The beads may be varied inshape and/or color to allow for a white cell differential to bereported. A variety of different shapes can be used, including spheres,raisin, raspberry, pear, peanut, snowman, oblong, or rod shaped. Beadsof different shapes and characteristics can be obtained, for example,from Bangs Laboratories, Inc., MAGSPHERE, and Microspheres-Nanospheres(a Corpuscular Company).

Referring to FIG. 3B, an illustrative set of beads for a surrogatecontrol material that can be used to perform CBC tests includes avariety of beads in which size, shape, and color are used to simulatevarious characteristics of cells, as presented in Table 1. In thefigure, images of the beads are shown in pairs with color microscopicimages on the left and corresponding fluorescent microscopic images onthe right. No fluorescent image is shown for the basophil and Red BloodCell (RBC) beads because these beads don't fluoresce. Image processingcan be used to determine the size, shape, bright-field (BF) color, andfluorescence (FL) color of the beads.

TABLE 1 Ref. Name Analog Size Shape BF color FL color 632 Red BloodCells RBC   5 μm round none none 634 Nucleated Red NRBC   5 μm roundnone green Blood Cells 636 Reticulocyte RET   5 μm round none red 620Neutrophil NEU   7 μm round none orange 622 Lymphocyte LYM   7 μm roundnone green 624 Monocyte MON   10 μm round none green 626 Eosinophil EOS7.25 μm raisin none green 628 Basophil BAS   7 μm round red none 630Immature Cell IC   10 μm round none orange 638 Platelet PLT   3 μm roundnone green

The beads in the surrogate control material presented above can beselected in such a way as to simulate a normal whole blood sample. Butthey can also be selected in ways that simulate conditions that simulatean abnormal whole blood sample. In one embodiment, the control materialis supplied in several lots with different count levels, including onenormal count, one low abnormal count, and one high abnormal count.

III. Calibration

Because the surrogate control material can be designed with a knownconcentration of beads, a count of these beads acts as a standard thatcan be used to derive one or more calibration values for the instrumentand/or cartridge. These values can be used to adjust one or more aspectsof measurements to be made on actual blood samples. One example of acount-based calibration value is a calibration factor that can adjustfor the actual metered volume of a sample. If actual metered volumes are5% smaller than expected, for example, count results can be multipliedby a corresponding factor to adjust for the discrepancy. Other aspectsof the measurement can also be adjusted for using factors, offsets,and/or other adjustment formulas. The dye 602 can be used in calibrationoperations, as well, such as to allow the analyzer to derive calibrationvalues for photometric measurements. Bead color and shape can also beused as known properties in deriving calibration values for the opticsor the camera.

Referring to FIG. 4, the cell analyzer 200 can be calibrated when it isfirst set up, and it can then be recalibrated periodically thereafter.An initial calibration run 700 can involve introducing a sample of thesurrogate control material 600 in the test cartridge 100 (step 702), andtaking measurements (step 704). The result can then be used to adjustthe operation of the analyzer or evaluated to determine whether anyadjustments to the analyzer are needed (steps 706-710).

IV. Ongoing Quality Control Operations

The cell analyzer 200 can perform ongoing operations to improve avariety of aspects of its operation, including the accuracy, precision,and reliability of its measurements. As noted above, these operationscan include performing calibration operations on a regular basis. Theycan also include ongoing monitoring of a variety of aspects of theanalyzer's operation.

Referring to FIG. 5, one type of quality control operation 740 is to usethe surrogate control material 600 to determine whether one or moreaspects of the cell analyzer 200 operates acceptably. This type ofoperation begins with providing a surrogate control material andintroducing it into the analyzer (steps 742, 744). Images of the mixtureare then acquired with the analyzer's microscope (step 746), and theanalyzer analyzes these images (step 748). A determination is then madeas to whether results of the analysis meet one or more predeterminedquality control thresholds (step 750). If the thresholds are met,patient samples can be processed. If they aren't met, remedial measuresmust be taken on the analyzer, such as performing adjustments to theanalyzer based on results of the determination, or by performing furthercalibration operations, or by performing technical service of theanalyzer.

Referring to FIG. 6, another type of quality control operation 760 is tomonitor the ongoing operation of the cell analyzer 200 as it processespatient samples. One way to do this is to capture and analyze images ofthe samples using the microscope and/or one or more monitoring cameras(step 762). Sample-specific information about properties of the patientsamples can be extracted from the captured images (step 764), and testedagainst predetermined standards to verify the operation of the analyzer(step 766).

Mean fluorescent intensity of cells can be monitored, for example, bycomparing the detected fluorescence level to a pre-determined acceptablelevel to detect potential defects in fluorescence measurements, such asdamage to the stain reagent or non-uniform staining. FIG. 7 shows themean fluorescence for each of eight illustrative sample runs with anacceptable excursion level for the mean White Blood Cell (WBC)fluorescence signal during a measurement performed by the cell analyzer200. When the mean fluorescence of the WBC falls below a pre-determinedminimum, the test, or a certain parameter of the test, is rejected. Inthis example, all of the WBCs in the test were used to determine meanfluorescence. However, a subset of WBCs or a specific cell class, suchas neutrophils, could be used to reject the test.

Referring to FIGS. 3C-E, roundness of red blood cells can be compared toa pre-determined set of sphering criteria to verify that the red bloodcells have been sphered correctly or detect if the sphering reagent wasdamaged or ineffective. This operation uses image processing to acceptor reject a sample based on pre-determined sphering criteria, includingroundness and secondary ring specifications. The image processingsoftware will determine a properly sphered cell 640 and pass if it meetsthese specifications. An incorrectly sphered RBC 642 will fail theroundness specification. Roundness can be quantified by comparing theperimeter of the object and its area to known mathematical formulae forvarious geometric shapes. An unsphered RBC 644 will fail the secondaryring specification. The detection of a secondary ring as shown in FIG.3E is due to the biconcave disc geometry of the unsphered RBC 644. Thesample can also be checked for clots, voids, and microbubbles, by use ofimage processing software and comparing the features of thesenon-cellular artifacts to pre-determined characteristics. The softwarecan also use image processing software to determine if the cells arecrowded or overlapping, which condition could cause an error incounting. Further examples of tests are presented below.

V. Discussion

Traditional CBC analyzers typically must be calibrated for white bloodcell count (WBC), red blood cell count (RBC), hemoglobin (Hgb), plateletcount (Plt), mean cell volume (MCV), and mean platelet volume (MPV). Allof these parameters are derived from the impedance part of the analyzer.There is generally no calibration for the flow cytometer, since it istypically only used to determine relative percentages, but not absolutecounts. However, if absolute counts are determined by the flow cytometerrather than using an impedance counter, then the flow cytometer shouldbe calibrated. Traditional CBC analyzers are typically calibrated everysix months, or when there has been a critical part replacement, or ifthe quality control checks identify that the system is inaccurate. Thecalibrators are similar in composition to stabilized blood controls,although they typically have an even shorter shelf life andopen-container use life. Traditional CBC analyzers are checked forcalibration, because protein or salt buildup and cellular debris cancause shifts in size measurements (MCV, MPV) and in absorbance for thehemoglobin photometer (Hgb), and because changes to flow rates ordilution ratios can cause changes to the absolute count results (WBC,RBC, and Plt).

As discussed above, the use of stabilized blood is a compromise toprovide a method of performing quality control. For years, this hasworked quite well for impedance and flow cytometry technologies, but itis not optimal for other technologies, such as microscopy.

Microscopy-based cell imaging and counting systems are subject todifferent calibration needs than traditional CBC systems. Where animaging system comprises an analyzer and a single-use test cartridge,the system can be factory calibrated and not require regularcalibration. The use of a single-use test cartridge eliminates thepossibility of carryover, blockages, or protein buildup. The absolutecount parameters—WBC, RBC, and Plt—are determined by the sample volumein the single-use disposable test cartridge metering valve. The cellsizing parameters—MCV and MPV—are measured and determined by the lensesand the pixel size of the camera, neither of which will generally changeover time. The hemoglobin photometer is calibrated at the factory and isnot usually at risk of drift because it maintains a dry interface withthe disposable test cartridge. The photometer can be calibrated using adye or film with a known absorbance value at the measurementwavelength(s) used for the hemoglobin measurement. The depth of thehemoglobin chamber in the cartridge is also a determining factor in theHgb result, which is controlled in the manufacturing process. The sameholds true with the with the test cartridge metering valve volume.

Traditional CBC analyzers use control material for all of the measuredparameters. Table 2 lists common error modes of traditional CBCanalyzers and how a stabilized blood control is used to help mitigatethe error. This is not an exhaustive list, but it does show the need fora control material with absolute counts, sizing parameters, and WBCdifferential in order to verify that traditional CBC systems are in goodworking condition.

TABLE 2 Common errors in traditional hematology analyzers and controlmethods to identify them. Error Mode Cause(s) QC Verification bloodcells settle operator fails to mix accuracy of RBC, WBC, plt sample tubeblood sampling and fluidics, metering accuracy of RBC, WBC, plt meteringmechanism dilution ratio fluidics accuracy of RBC, WBC, plt RBC spheringreagent integrity, accuracy of RBC, MCV fluidics RBC lysing reagentintegrity, accuracy of WBC, Hgb fluidics Hgb reagent reagent integrity,accuracy of Hgb (species conversion) fluidics Hgb calibration fluidics,cleanliness of accuracy of Hgb Hgb chamber cell size calibrationfluidics, cleanliness of accuracy of MCV, MPV counting chambers cellcount fluidics, cleanliness of accuracy of RBC, WBC, plt calibrationcounting chambers WBC differential fluidics, cleanliness of accuracy of3-part WBC (impedance) counting chambers, Diff (GRN %, LYM %, partialblock MON %) WBC differential fluidics, cleanliness of accuracy of5-part WBC (flow cytometry) flow cytometer, partial Diff (NEU %, LYM %,block MON %, EOS %, BAS %)

Proposed is a combination of a surrogate blood control and a system ofinternal software checks that can be utilized to ensure accurateperformance of microscopy-based cell imaging and counting systems. Thequality control material is supplied in three levels, similar to thosethat would be used with blood-based controls. The material consists of adye and beads of various sizes, shapes, and/or colors. The dye orplurality of dyes is chosen such that the concentration and absorbancesimulates whole blood at least at one wavelength of light. The beads maybe silica, polystyrene, or other polymer in composition. The sizes ofthe beads are typically in the range of 1-20 micrometers to simulate thesize of platelets, red blood cells, reticulocytes, and the differentwhite blood cells. Some or all of the beads may fluoresce to simulateWBCs, platelets, or reticulocytes. The beads may be varied in shapeand/or color to simulate a white cell differential to be reported.

When used in conjunction with a single-use test cartridge, a drop ofquality control material is deposited by the user into a test cartridgein the same way that a patient sample is deposited. No special qualitycontrol test cartridge is required. The quality control material isanalyzed in the cartridge with the same process as a patient sample.

When the image-based system does not use a single-use cartridge, or ifthe test cartridge does not contain all of the fluidics needed for thetest, the analyzer can aspirate the QC sample, in the same way that itaspirates a whole blood sample. The use of a surrogate control materialcan be particularly useful in systems, where the fluidics are part of ananalyzer.

Table 3 is a list of the error modes that could arise in performing aCBC test. Two methodologies are presented: one using a whole bloodcontrol (if one existed that could meet the need for microscopy) and theother using the new control comprising of a daily surrogate controlmaterial and internal software quality controls. The use of softwarecontrols on every sample is particularly important for single-use testdevices.

TABLE 3 Failure modes on a Microscopy-Based Cell Imaging and CountingSystem with a single-use test cartridge and how surrogate controls andsoftware controls are used to identify errors. Whole Blood Control NewControl Error Mode Cause(s) Methodology Methodology blood cells settleoperator fails to if not mixed, cells if not mixed, beads settle in mixsample tube settle in tube tube blood cells settle operator fails tocells settle in sample beads settle in sample input start test within 1input minute blood sample consumable failure accuracy of measuredaccuracy of measured metering absolute counts of absolute counts ofbeads cells metering pneumatics failure accuracy of measured accuracy ofmeasured diluent/stain absolute counts of absolute counts of beads cellsmixing blood consumable failure cell distribution bead distribution anddiluent transfer of mixed pneumatics failure cell distribution beaddistribution sample to imaging region homogeneity of pneumatics failurecell distribution bead distribution sample in imaging region WBC countreagent integrity, cells fluoresce and beads fluoresce and can beconsumable failure can be counted counted WBC differential reagentintegrity, WBC sub- monitor mean fluorescence consumable failurepopulations stain on every sample differently, creating a differentialRBC count reagent integrity, cells are counted in beads are counted inconsumable failure bright-field bright-field MCV reagent integrity RBCare sphered and monitor cell sphering on measurement measured everysample, measure size of beads platelet counting reagent integrity,platelets fluoresce beads fluoresce and can be consumable failure andcan be counted counted hemoglobin reagent integrity, reagent-free,reagent-free, measured on measurement consumable failure measured on dyeof control hemoglobin of control

Traditional CBC analyzers generally use stabilized whole blood controlmaterials to perform a QC check on the white cell differential. For athree-part differential using impedancemetry, any drift or partial clogin the impedance channel could cause erroneous results in the three partdifferential. Similarly for flow cytometers, a partial occlusion in theinjector nozzle to the flow cell can cause the stream of cells to be offcenter, thus causing erroneous results in the five-part differential. tobe In a system that does not have fluidics or possible mechanicalfailures, the primary cause for a failed WBC differential would be afailure of the stain reagent. Monitoring the mean fluorescence on everysample can identify a problem with reagent or stain integrity on everycartridge. This is preferable to using quality control material toperform a QC check once per day.

The control material can also be used to check operator proficiency.Before a blood sample is run on a CBC analyzer, it should be properlymixed by the operator. If the operator fails to mix the blood sample,the analyzer could generate an erroneous result. QC material can be usedto verify operator proficiency in mixing of the blood sample, as thebeads will settle in the same manner that blood cells settle, andgenerate an erroneous result.

A set of internal process and system controls that are performed onevery sample using quality control cameras can be used to furthercontrol cell imaging and counting systems. The cameras ensure that everypart of sample preparation is performed correctly for every test. Table4 details the procedural controls.

All operational steps—including sample metering, dilution, analysis, andanalyzer self-checks are handled and controlled automatically within thedevice, without the need for user intervention. The analyzer performsself-checks during initialization to ensure that the system is workingproperly. These self-checks include the processors, the cameras, thesafety interlocks, the microscope stage, and the diluter mechanism.

The single-use test cartridges are managed through barcode intelligence.Embedded within the barcodes are the lot number, the expiration date,and the serialization for each cartridge. When the cartridge isinserted, the analyzer reads the barcode automatically, eliminating thepossibility of using an invalid cartridge, using an expired cartridge,or using a test cartridge more than once. Intelligence can also bemanaged by use of RFID tags, 1-Wire, iButton, EEPROM, or similardevices.

The system uses one or more quality control cameras for comprehensivemonitoring of all sample processing steps. In the example of using twocameras, one quality control camera is used to verify that the meteredblood sample is free from bubbles and that the metering process isaccurate, without any loss of sample. This ensures that the meteredvolume of blood in every sample is accurate. This same quality controlcamera ensures that the entire sample from the mixing chamber has beenemptied into the imaging chamber at the completion of the dilution step.The second quality control camera views the entire imaging chamber ofthe cartridge. This allows for the verification of the dilutionintegrity. If there are any bubbles or voids in the channel, thesoftware automatically masks these sections and removes them from thecalculation. The combination of these two cameras ensures that themetering and dilution on every cartridge is completely controlled andverified.

A third camera is part of the microscope and is used for analyzing theimages of the cells at 20× magnification. In addition to the cellcounting, sizing, and classification, this camera also verifies sampleintegrity at a microscopic level. For example, the software checks theimages for clots and microbubbles and for overlapping cells. If thecamera detects a problem with sample integrity, the analyzer will rejectthe sample. A different magnification could be used to analyze thequality of the cells and the fluid matrix.

TABLE 4 Procedural failure modes on the Microscopy-based Cell Imagingand Counting System and internal software checks are implemented asverification or cause for rejection. Error Mode Software Process Controlinsufficient blood sample QC camera verification, reject air bubbles inblood sample QC camera verification, reject test slide loadingmechanical design for fail-safe insertion of the test slide meteringblood sample QC camera verification metering diluent/stain controlled byedge detectors, QC camera verification washout of valve QC cameraverification mixing blood and diluent cell distribution, QC cameraverification transfer of mixed sample to imaging cell distribution, QCcamera region verification homogeneity of sample in imaging celldistribution, QC camera region verification determine percent samplingof the use QC camera to verify area whole voids or bubbles in theimaging use QC camera to adjust area chamber clots or microbubble in theimaging use microscope camera to detect chamber overlapping cells usemicroscope camera to detect cartridge reuse or misuse unique barcodedserial # for each cartridge reagent quality stability tracking bybarcode

Some or all of the various quality control and calibration tasksperformed by the analyzer can be carried out using a speciallyprogrammed general purpose computer, dedicated hardware, or acombination of both. These can be incorporated into the analyzer as partof the system controller 250 and/or image processor/computer 290. Someor all of the control, image processing, calibration, and otherfunctionality can also be provided through software and/or hardwarelogic provided by a standalone processing system located proximate thesystem. And parts of the functionality can even be provided from aremote location through a public or private communication network. Inone embodiment, the system is based on stored software instructionsrunning on a Microsoft Windows®-based computer system, but otherplatforms could be used as well, such as Android®-, Apple®-, Linux®-, orUNIX®-based platforms.

The present invention has now been described in connection with a numberof specific embodiments thereof. However, numerous modifications whichare contemplated as falling within the scope of the present inventionshould now be apparent to those skilled in the art. For example, whilemany of the techniques and materials described in this application areparticularly well suited to use with microscopy-based analyzers, many ofthem may also be applied to quality control and calibration oftraditional types of CBC analyzers. Therefore, it is intended that thescope of the present invention be limited only by the scope of theclaims appended hereto. In addition, the order of presentation of theclaims should not be construed to limit the scope of any particular termin the claims.

What is claimed is: 1-40. (canceled)
 41. A method of verifying theoperation of a cell analyzer having an automated microscope and imageprocessing software for counting and analyzing cells and platelets inblood and which utilizes a test cartridge having a diluent/and or stain,the method comprising: a) introducing into the test cartridge a mixtureof 1) a plurality of a first type of microbeads in a known amount ofsolution having a first known characteristic and a concentration thatsimulates a concentration of a first type of cells in blood and 2) asecond plurality of micro-beads in a known amount of solution having asecond known characteristic and concentration that simulates aconcentration of a second type of cells in blood; b) interfacing thetest cartridge with the cell analyzer; c) separating a known amount ofthe mixture of micro-beads from the remaining amount of the mixture ofmicro-beads in the test cartridge; d) mixing the known amount of themixture of micro-beads in the test cartridge with an amount of diluentand/or stain that is sufficient to form a substantially uniform mixtureof micro-beads and diluent and/or stain, wherein the micro-beads aresuspended therein; e) transferring the entire mixture into the imagingchamber that is defined in the body of the test cartridge; f) capturingone or more digital images of the mixture in the imaging chamber thatare selected to be statistically representative of the number anddistribution of the first and second types of microbeads; g) countingall of the at least one type of micro-bead in the images using imagingprocessing software; h) obtaining a number of micro beads per unitvolume of at least one type of micro-beads in the mixture; and i)comparing the number of micro-beads per unit volume of the one type ofbeads in the imaging chamber with the concentration of the one type ofbeads in solution.
 42. The method of claim 41 wherein the mixture ofbeads is counted by image processing logic associated with the analyzer.43. The method of claim 41 wherein the two types of micro-beads arecounted separately by image processing logic associated with theanalyzer according to their different characteristics.
 44. The method ofclaim 41 wherein one type of micro-bead is a fluorescent micro-bead. 45.The method of claim 41 wherein the providing provides a mixture ofmicro-beads of different sizes.